The objective of the research proposal is to obtain a molecular understanding of the phosphomannosyl targeting system which functions to deliver newly synthesized acid hydrolases to lysosomes. Defects in this intracellular protein transport pathway give rise to severe lysosomal storage diseases. The specific aims include: (1) The characterization of the protein recognition domain on DNase I that is necessary for interaction with phosphotransferase which catalyzes the first step in the generation of the Man-6-P recognition marker. We will use site- directed mutagenesis to identify residues on the surface of DNase I that determine binding to phosphotransferase and analyze how these residues direct phosphorylation at selected glycosylation sites. (2) To complete the cloning of phosphodiester alpha-GlcNAcase and define the protein domains that determine its Golgi localization. This enzymes removes the covering GlcNAc from acid hydrolase oligosaccharides to expose Man-6-P residues essential for their binding to Man-6-p receptors. cDNA clones encoding wild-type and mutant enzyme will be transfected into COS cells and analyzed for Golgi localization and trafficking using pulse-chase experiments and E/M immunogold localization. (3) To investigate the mechanism whereby binding of acid hydrolases (but not IGF-II) to the Man-6-P/IGF-II receptor accelerates the rate of internalization via clathrin-coated pits. (4) To identify the Golgi membrane docking protein(s) that interacts with the heterotrimeric AP-1 adaptor complex and serves as the nucleation site for the subsequent assembly of Clathrin-coated vesicles on the trans-Golgi network. These vesicles carry the Man-6-P receptor.ligand complex on the first leg of its intracellular itinerary. We have shown that the initial event in coat assembly involves the ADP-ribosylation factor mediated recruitment of AP-1 from the cytosol. The assembled complex on the Golgi membrane will be solubilized with mild detergent and purified by gel filtration and affinity chromatography. Once the docking protein(s) has been identified and cloned, we will use molecular techniques to define the structural determinants that specificity its intracellular localization and dissect the protein-protein interactions that underlie AP-1 recruitment. This study is designed to provide a detailed understanding of the nature and function of the proteins responsible for clathrin-coated vesicle formation on Golgi membranes.